Reduced cerebral blood flow can cause a range of problems from light headaches to severe brain damage and death. There are many situations that can lead to reduced cerebral blood flow. Examples of such situations include hemorrhagic stroke, brain injuries, occlusive stroke, cerebral vasospasm and hypotension.
Subarachnoid hemorrhage (SAH) accounts for 5-10% of all strokes. The incidence is 2-20 events per 100,000 population per year with a case fatality rate of 20-60%. Ingall, T et al., Stroke 31: 1054-1061 (2000); Stroke 31: 1843-1850 (2000). SAH occurs most commonly after rupture of cerebral aneurysms or head trauma leading to subarachnoid bleeding and clot formation. After the bleeding, cerebral blood flow initially falls due to an elevated cerebrospinal fluid pressure that reduces effective perfusion pressure combined with the release of vasoactive agents from the blood that increases cerebral vascular tone. This initial phase of injury following SAH is associated with a high mortality (30-50%). Later cerebrovasospasm (CVS) develops with a delayed onset (2-3 days in rats, 5-7 days in human beings). The mortality associated with this delayed CVS is even more devastating and approaches 70%. Weir, B., Br. J. Neurosurg 9: 375-390 (1995). Very little is known about the factors that are released by blood and reduce cerebral blood flow in the initial phase after SAH. Even less is known about what mediates delayed CVS. Previous studies have indicated that the delayed CVS following SAH is associated with activation of protein kinase C (PKC), diminished activity of K+ channels and depolarization of vascular smooth muscle (VSM) cells. Depolarization of cerebral VSM cells increases influx of calcium which potentiates the vasoconstrictor response to endothelin (ET), thromboxane, serotonin and other vasoconstrictors produced by clotting blood and diminishes the responsiveness of cerebral vessels to endogenously formed vasodilators such as nitric oxide (NO). The impaired response to NO has been postulated to be due to the binding of NO to free hemoglobin after hemolysis of erythrocytes, Edwards, D. H. et al., J. Neurosurg. 76: 830-837 (1992), or increased degradation of NO by superoxide formed by oxidation of hemoglobin. Misra, H. P. et al., J. Biol. Chem. 247: 6960-6962 (1972); Winterbourn C C et al., Biochem. J. 155: 493-502 (1976). Moreover, there is evidence that the activity of one of the second messengers of NO, soluble guanylyl cyclase, is reduced following SAH. Faraci, F. M. and Sobey, G. C., Brain Res. 821: 368-373 (1999); Sehba, F. A. et al., Stroke 30: 1955-1961 (1999).
Other studies have explored the role of upregulation of vasoconstrictor pathways in mediating CVS following SAH. ET levels increase after SAH. The initial fall in cerebral blood flow observed two hours after induction of SAH in rats is attenuated by inhibitors of the synthesis of ET and by ET receptor blockers. Clozel, M. and Watanabe, H., Life Sci. 52: 825-834 (1993). Enhanced fatty acid turnover and increased formation of vasoconstrictor metabolites of arachidonic acid (AA) have also been observed following SAH. Juvela S, J. Neurosurg. 92: 390-400 (2000); Seifert, V. et al., J. Neurosurg. 82: 55-62 (1995). Recent studies have indicated that 20-hydroxyeicosetetraenoic acid (20-HETE) is a metabolite of AA produced in the cerebral circulation. However, the role of 20-HETE in the pathogenesis of CVS following SAH is unknown. 20-HETE is produced by enzymes of the cytochrome P450 (CYP) 4A family that are expressed in VSM cells in cerebral arteries. Harder, D. R. et al., Am J Physiol Heart Circ Physiol. 266: H2098-H2107 (1994); Gebremendin, D. et al., Circ Res. 87: 60-65, 2000. The CYP4A family members for 20-HETE formation include CYP4A1, 2, 3 and 8 in rats, CYP4A10, 12 and 14 in mice, and CYP4A11 in human. Enzymes of the CYP4F family are also involved in 20-HETE production. The CYP4F family members for 20-HETE formation include CYP4F1, 4, 5 and 6 in rats, and CYP4F2 and 3 in human. CYP4F2 has been shown to produce 20-HETE when incubated with AA. Powell, P. K. et al., J Pharmacol Exp Therap 285: 1327-1336, 1998. CYP4F3 has been shown to be expressed in polymorphonuclear white blood cells that produce 20-HETE. Bednar, M. M. et al., Biochem Pharmacol 60: 447-455, 2000; Rosolowsky, M. et al., Biochem Biophys Acta 1300: 143-150, 1996.
20-HETE is a potent constrictor of cerebral arteries (EC50<10 nM). 20-HETE activates PKC and depolarizes VSM cells by inhibiting the large conductance KCa channel. 20-HETE also increases Ca2+ influx via L-type Ca2+ channels in the cerebral vasculature, Harder, D. R. et al., Am J Physiol Heart Circ Physiol. 266: H2098-H2107 (1994); Gebremedhin, D. et al., J. Physiol (Lond). 507 (Pt 3): 771-781 (1998), and plays a critical role in the mechanism underlying the autoregulation of cerebral blood flow in rats. Alonso-Galicia, M. et al., Stroke 30: 2727-2734 (1999); Gebremedhin, D. et al., Circ. Res. 87: 60-65 (2000). Vasoconstrictor peptides like angiotensin II and ET stimulate the formation of 20-HETE. Oyekan, A. et al., Am J Physiol Regulatory Integrative Comp Physiol. 273: R293-R300 (1997); Croft, K. D. et al., Am. J. Physiol Renal Physiol. 279: F544-F551 (2000). The vasodilator NO inhibits the formation of 20-HETE. Alonso-Galicia, M. et al., Stroke 30: 2727-2734 (1999). Activated polymorphonuclear leukocytes (PMN) and cerebral arteries avidly produce 20-HETE. Harder, D. R. et al., Am J Physiol Heart Circ Physiol. 266: H2098-H2107 (1994); Gebremedhin, D. et al., J. Physiol (Lond). 507 (Pt 3): 771-781 (1998); Alonso-Galicia M. et al., Stroke 30: 2727-2734 (1999); Gebremedhin, D. et al., Circ. Res. 87: 60-65 (2000); Bednar, M. M. et al., Biochem. Pharmacol. 60: 447-455 (2000); Rosolowsky, M. et al., Biochem Biophys Acta 1300: 143-150, 1996; Lange, A. et al., J. Biol. Chem. 272: 27345-27352 (1997). However, it is not known whether treating an animal with a 20-HETE synthesizing enzyme inhibitor will prevent the initial fall in cerebral blood flow associated with SAH (hemorrhagic stroke) or the delayed CVS and cerebral ischemia. Moreover, drugs that are currently available for inhibiting the formation of 20-HETE have serious limitations that greatly restrict their potential as therapeutic agents. For example, although 17-ODYA inhibits the synthesis of 20-HETE, it is not a specific inhibitor because it is equally effective at blocking the formation of EETs. Zou, A. P. et al., J Pharmacol Exp Therap 268: 474-481, 1994. EETs are potent vasodilators formed in the brain. Blockade of the synthesis of EETs may oppose any beneficial effects associated with blockade of the formation of 20-HETE. Alkayed, N. J. et al., Am J Physiol Heart Circ Physiol 271: H1541-H1546, 1996; Gebremendhin, D. et al., Am J Physiol Heart Circ Physiol 263: H519-H525, 1992. 17-Octadecynoic acid (17-ODYA) also binds to plasma proteins and does not cross the blood brain barrier when given systemically. DDMS is a more specific inhibitor of the formation of 20-HETE. Alonso-Galicia, M. et al., Hypertension 29:320-325, 1997; Wang, M. H. et al., J Pharin Exp Therap 284:966-973, 1998. However, it too binds to plasma proteins and does not cross the blood-brain barrier when given systemically.
The effects of intracerebroventricular injection of DDMS on cerebral blood flow has been examined in previous studies. DDMS has no effect on baseline cerebral blood flow. However, it blocks autoregulation of cerebral blood flow, i.e., constriction of cerebral vessels associated with elevations in cerebral blood flow. Gebremendin, D. et al., Circ Res. 87:60-65, 2000. DDMS has also been reported to prevent the rise in cerebral blood flow produced by the NO donor, DEA NONOate. Alonso-Galicia, M. et al., Stroke 30:2727-2734, 1999. However, there is no in vivo information available as to whether DDMS or 17-ODYA have beneficial effects in preventing or reversing a fall in cerebral blood flow such as that associated with SAH or stroke and other cerebral vascular diseases.